The zebrafish is a superb genetic system for studying vertebrate development and modeling human diseases. Several large-scale mutagenesis screens have generated thousands of mutant fish with defects in important biological processes. However, this system still lacks efficient ways to perform reverse genetics and gene tagged mutagenesis. Recently, we have shown that normal zebrafish individuals can be cloned by nuclear transfer using donor nuclei from long-term cultured cells. In this proposal, we will explore the utility of this finding in two areas. First, we will establish a gene trap procedure by which cultured cells will be selected for gene trap events and then used to produce zebrafish by animal cloning. Second, we will develop targeted mutagenesis methods for reverse genetics in zebrafish. A number of selected candidate genes will be disrupted by homologous recombination in cultured fibroblast cells and the nuclei carrying the homologous recombination events will then be used to produce cloned zebrafish. For the past three years, our laboratory has devoted significant resources and time towards developing gene-targeting technology in zebrafish. A group of scientists with unique technological talents have been assembled. With a continuous support, we believe that we can achieve the proposed goals and zebrafish will soon offer all the essential tools that have been enjoyed by the mouse research community. The two systems will then truly complement each other to advance our understanding of vertebrate developmental biology.